Medium for preserving biological materials

ABSTRACT

The present invention relates to a medium allowing the preservation and cryopreservation of biological materials such as animal cells and viral particles that are directly injectable or reinjectable into an organism. A medium for preserving and/or freezing biological materials, including a saline solution, modified fluid gelatin and human serum albumin, is disclosed.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a §371 national phase filing of InternationalApplication No. PCT/FR97/00385, filed Mar. 5, 1997. This applicationclaims the benefit of foreign priority under 35 U.S.C. §119(a) ofapplication FR96/03074, filed Mar. 12, 1996 in France.

BACKGROUND OF THE INVENTION

The present invention relates to a medium allowing the preservation andcryopreservation of biological materials such as animal cells and viralparticles, which is directly injectable or reinjectable into anorganism. It relates more particularly to a medium for the preservationof biological material comprising a saline solution, modified fluidgelatin and human serum albumin.

In cell therapy or gene therapy as well as in blood transfusion or bonemarrow transplantation, one of the principal problems encountered isthat of the preservation of biological material. It is indeed importantto be able to preserve biological material, under good conditions ofviability, for a sufficiently long period of time compatible withindustrial scale production and storage and also to make it possible tocarry out certain tests. The most commonly used method of preservationconsists in freezing the said material. However, during the freezing ofcells, for example, they undergo a very high stress due in particular toa phenomenon of exosmosis and to the formation of ice inside the cell.These phenomena cause numerous cell lyses both during freezing andduring thawing. The capacity to divide or to differentiate of even thenon-lysed cells may be irreversibly impaired. It is very important to beable to obtain a high viability level after thawing for cells which haveto be injected into a living organism (blood transfusion, bone marrowtransplantation or cell therapy for example), it is indeed futile toreinject dead or damaged cells. Likewise, when cells have to berecultured, it is equally important that the viability level is high. Upuntil now, a cryoprotectant which prevented cell lysis was added to themedium. The protecting agent most widely used and which gives the bestresults is a non-injectable preservative, dimethyl sulphoxide or DMSO.DMSO becomes inserted into the cell membranes and makes it possible tostabilize them. It thus prevents the destruction of the cells. A systemsuch as this makes it possible, at the time of thawing, to have a largenumber of live and viable cells which are capable of dividing. However,this method poses a major problem when the said cells have to beintroduced or reintroduced into an organism. Indeed, DMSO is a productwhich is toxic to the cells at room temperature. DMSO permeabilizes themembranes, causing the death of the cell. It should therefore be removedbefore being able to reimplant the cells in the case of an autograft ofmodified cells or to implant them in the case of a heterograft, forexample, of bone marrow cells or alternatively to carry out transfusionin the case of blood cells. The elimination of the DMSO is achieved,after thawing, generally by diluting the sample in ten volumes ofDMSO-free medium, followed by centrifugation and elimination of thesupernatant. This operation is repeated several times until practicallyall the DMSO is eliminated. This treatment involves a loss of time and,by multiplying the manipulations, increases the risks of contaminationby external pathogenic agents and of losses of the said biologicalmaterial.

In the presence of DMSO, the percentage of viable cells after thawing isgreater than seventy per cent under optimum freezing/thawing conditionsas defined later. Without DMSO, this percentage is less than twenty percent. Up until now, freezing in the presence of DMSO was the mosteffective and therefore the most widely used method.

BRIEF SUMMARY OF THE INVENTION

The applicant investigated a new type of medium which makes it possibleto avoid manipulations subsequent to the thawing while maintaining ahigh percentage of viable cells. For that, the applicant developed afreezing/preservation medium which makes it possible to obtain a highviability on thawing without using DMSO or another cytotoxiccryopreservative. The advantage of such a medium stems from the factthat the solution is injectable immediately after the thawing withoutany manipulation being necessary. It then becomes possible to carry outthe thawing directly in the operating theatre, thereby reducing the timebetween the thawing and use, which also makes it possible to remainconstantly in a sterile medium and therefore to reduce to a minimum therisks of external contaminations.

A first subject of the invention relates to a medium for thepreservation and/or freezing of biological material comprising a salinesolution, modified fluid gelatin and human serum albumin (HSA).

As indicated above, this medium lacks any toxic agent and may beadministered directly to an organism. It may be used for preserving,optionally in frozen form, various biological materials such as viruses,cells, platelets and the like.

DETAILED DESCRIPTION OF THE INVENTION

The first element entering into the composition of the medium accordingto the invention is the saline solution. The saline solution is moreparticularly a solution which is isotonic with the plasma. The saltsentering into the composition of this solution may vary. Advantageously,it comprises chlorides, such as sodium chloride, potassium chloride,calcium chloride and/or magnesium chloride, and lactates, such as, forexample, sodium lactate. More particularly, the isotonic saline solutiongenerally comprises sodium chloride, potassium chloride, magnesiumchloride and sodium lactate. According to another variant, magnesiumchloride is replaced by calcium chloride. In this case, the saltconcentrations of the saline solution are equivalent or practicallyequivalent to those of a “Ringer-lactate” solution. Such a solution isusually used in perfusion to compensate for a dehydration or a loss ofphysiological saline for example.

According to a specific embodiment of the invention, the saline solutionis essentially composed of NaCl, MgCl₂, KCl and lactate whose respectivefinal concentrations in the medium are given in Table 1 below.

TABLE 1 Minimum Maximum concentration concentration Specific Salt (g/l)(g/l) example NaCl 2.0 9 5.7 MgCl₂ 0.05 0.2 0.093 KCl 0.05 0.5 0.247Lactate 0.5 4 2.25

Gelatin is the second constituent entering into the composition of themedium according to the invention. It is a protein composed of variousamino acids linked by adjacent amino and carbonyl groups, so as to givethe conventional peptide bond. The molecular weight of gelatin ischaracteristic and high (the average molecular weight values vary fromabout 10,000 to 100,000) and it is substantially heterogeneous for agiven gelatin type or quality. Gelatin is composed of rod or asymmetrictype molecules resulting from the hydrolysis of the long chains of thepolypeptide residues in the white connective tissue. Experience hasshown that the primary hydrolysis of collagen occurs at intervals atreactive sites of these chains to produce a non-degraded related idealgelatin molecule. This continues to a varying degree in a secondaryhydrolysis at random intervals on the less reactive bonds of the idealgelatin molecule. This explains how the degradation reaction isresponsible for the random heterogenous molecular pattern of a specificgelatin sample. Likewise, each protein constituting the gelatin has adefined isoelectric point at which the ionization and, consequently, thephysical and chemical reactivity is minimal. These properties areespecially the solubility, the viscosity and the colloidal osmoticpressure. The asymmetry of the gelatin molecule therefore gives, withthe heterogenous molecular pattern, intrinsic properties of gelformation and viscosity to the gelatin solutions prepared for the mediumaccording to the invention.

The gelatin itself (sterilized and freed from pyrogenic and antigenicsubstances) has already been used as product for replacing blood plasma,but it has raised a number of problems, in particular for itspreservation, because it gels at room temperature. This has led to othercompounds derived from gelatin, generally designated by the termmodified fluid gelatin, which make it possible to overcome thesedisadvantages in particular.

Among the modified fluid gelatins, there may be mentioned for exampleoxypolygelatin, obtained by polymerization of gelatin with glyoxal andoxidation with H₂O₂. Other modified fluid gelatins are obtained byreacting gelatin (preferably having a molecular weight range of about15,000 to 36,000) with succinic, citraconic, itaconic, aconitic ormaleic anhydride or succinyl or fumaryl chloride, as described in Frenchpatent No. 1,291,502. All these gelatin derivatives are compatible witha pharmaceutical use and may be introduced into the blood streamdirectly in an isotonic saline solution. Modified fluid gelatins havealso been described in patents U.S. Pat. Nos. 2,525,753, 2,827,419 and3,108,995.

More generally, the modified fluid gelatins according to the inventionconsist of chemically modified collagen hydrolysis products which arecompatible with a pharmaceutical use. They are preferably productshaving an average molecular weight of between 10 kD and 100 kD, andstill more preferably between 15 kD and 40 kD. They are preferablymodified by reacting with an anhydride, so as to obtain a final product,having a fluidity adapted to the desired use, according to theteachings, for example, of patent FR 1,291,502. This is preferablysuccinic, citraconic, itaconic, aconitic or maleic anhydride. Aparticularly advantageous modified fluid gelatin consists of thehydrolysis product of collagen having an average molecular weight ofbetween 15 kD and 40 kD, modified by reacting with succinic anhydride.The modified fluid gelatins according to the invention may be preparedby the techniques of persons skilled in the art, which are especiallydescribed in the abovementioned patents.

The third element entering into the composition of the medium accordingto the invention is human serum albumin. Human serum albumin (HSA) is anon-glycosylated monomeric protein of 585 amino acids, with a molecularweight of 66 kD. Its globular structure is maintained by 17 disulphidebridges which create a sequential series of 9 double loops (Brown J. R.,“Albumin Structure, Function and Uses”, Rosenoer, V. M. et al. (eds.)Pergamon Press, Oxford (1977) 27-51). The genes encoding HSA are knownto be highly polymorphic, and more than 30 apparently different geneticvariants have been identified by electrophoretic analysis under variedconditions (Weitkamp, L. R. et al., Ann. Hum. Genet. 37 (1973) 219-226).The HSA gene is cut in 15 exons by 14 intron sequences and comprises16,961 nucleotides, from the supposed “capping” site up to the firstsite of addition of poly(A).

Human albumin is synthesized in the hepatocytes of the liver, and thensecreted into the blood flow. This synthesis leads, in a first instance,to a precursor, prepro-HSA, which contains a signal sequence of 18 aminoacids directing the nascent polypeptide in the secretory pathway.

HSA is the most abundant blood protein, with a concentration of about 40g per litre of serum. There are therefore about 160 g of circulatingalbumin in the human body at any time. The most important role of HSA isto maintain a normal osmolarity of the blood flow. It also has anexceptional binding capacity for various substances and plays a roleboth in the endogenous transport of hydrophobic molecules (such assteroids and bile salts) and in that of different therapeutic substanceswhich may thus be transported to their respective sites of action.Furthermore HSA has been recently implicated in the breakdown of theprostaglandins.

The HSA used within the framework of the present invention may be eitherof natural origin (purified HSA) or of recombinant origin (rHSA).

In this regard, the natural HSA is generally produced by purificationfrom biological material of human origin. In particular, it is obtainedby conventional techniques for fractionation of plasma obtained fromblood donations (Cohn et al., J. Am. Chem. Soc. 68 (1946) 459 pp), or byextraction from the human placenta, according to the technique describedby J. Liautaud et al. (13th International IABS Conference, Budapest; A:“Purification of proteins. Development of biological standard”, Karger(ed.), Bale, 27 (1973) 107 pp). Preferably, the purified albumin usedwithin the framework of the present invention is a plasma albumin. Mostparticularly, a commercial plasma albumin solution may be used.

The development of genetic engineering and of new extraction andpurification techniques has opened the possibility of obtaining, at alower cost price, improved products of higher purity, of greaterstability and without risk of viral contamination (for example hepatitisB and AIDS). Given the importance of the HSA market, the possibility ofproducing this protein by a recombinant route has been widely studied.Thus, numerous expression systems have been studied for the preparationof the recombinant HSA.

More particularly, as regards the bacterial hosts, the first geneticengineering experiments used the bacterium E. coli as host organism.Thus, European patents EP 236 210, EP 200 590, EP 198 745, or EP 1 929describe processes for the production of HSA in E. coli using differentexpression vectors, different transcriptional promoters, and differentsecretory signals. Subsequently, studies relating to the secretion ofHSA in Bacillus subtilis were also carried out (Saunders et al., J.Bacteriol. 169 (1987) 2917). As regards the eukaryotic hosts, processesfor the production of HSA were developed using yeasts as host organism.Thus, it has been possible to demonstrate the production of HSA underthe control of the chelatin promoter in S. cerevisiae (Etcheverry etal., Bio/Technology 4 (1986) 726). The production of HSA has also beenmentioned in the brewery yeast during the manufacture of beer, using apost-fermentative process (EP 201 239). More recently, patentapplication EP 361 991 describes a particularly efficient system usingthe yeast Kluyveromyces as host organism, transformed with vectorsderived from the plasmid pKD1. Particularly high levels of HSA secretedinto the culture medium were able to be obtained with this system.Finally, the production of recombinant HSA has also been described inPichia pastoris, (EP 344 459). In addition, the purification of HSA hasalso been the subject of numerous studies (EP 319 067).

A recombinant or natural HSA is advantageously used which meets certainquality criteria (homogenetic, purity, stability). Thus, thepharmacopoeia sets a number of parameters for the plasma albuminsolutions, namely a pH value, a protein content, a polymer and aggregatecontent, an alkaline phosphatase content and a certain proteincomposition. It imposes, furthermore, a certain absorbance, thecompliance with a test of sterility, with a test of pyrogens and oftoxicity (see “Albumini humani solutio” European Pharmacopoeia (1984)255). The use of an albumin corresponding to these criteria, althoughnot essential, is particularly preferred.

Advantageously, the compositions according to the invention comprise apurified human plasma albumin or a recombinant human albumin, preferablyproduced in an eukaryotic host. In addition, the term HSA comprises, forthe purposes of the invention, any natural variant of human albumin,resulting from the polymorphism of this protein. It is also possible touse an HSA equivalent, that is to say any HSA derivative conserving theproperties of HSA. These derivatives may be especially N-terminalfragments of HSA.

The media according to the invention may be prepared in various ways.The different components may be mixed together, and then the biologicalmaterial added to the mixture. It is also possible to mix one or twocomponents with the biological material and then to add the last or thelast two component(s). Preferably, a medium comprising the threecomponents is prepared, to which the biological material is then added.The preparation of the medium and the addition of biological materialare performed under sterile conditions. According to a specificembodiment, the modified fluid gelatin is added to a saline solution,and then the HSA is added to the medium. In this regard, a preferredembodiment consists in using a specific mixture having the samecomposition as a blood plasma substitute, Plasmion (patent FR2,042,381), to which the HSA is then added. Plasmion is a commercialsolution composed of a saline solution and modified fluid gelatin. Itscomposition is the following:

Modified fluid gelatin 30 g/l Sodium chloride 5.382 g/l Magnesiumchloride 0.143 g/l Potassium chloride 0.373 g/l Sodium lactate 3.360 g/lWater for injection qs 1000 ml

Plasmion is usually used as vascular filling solution in the restorationof the circulating blood volume, or for a haemodilution with a reductionin blood viscosity and an increase in the microcirculation, oralternatively for the restoration of the ionic equilibrium and theprevention of acidosis. A preferred subject of the present inventionrelates to a medium allowing the preservation and/or freezing ofbiological material in which plasmion serves as mixture of salinesolution and modified fluid gelatin. Such a medium comprises plasmionand human serum albumin.

The respective proportions of the components of the media according tothe invention may be adapted by persons skilled in the art according tothe biological material considered. As illustrated in the examples,although certain concentration ranges are preferred, the proportions maybe modified. In this regard, the preferred salt concentration intervalswere presented in Table 1 above. As regards the gelatin and the serumalbumin, they vary preferably in an albumin/gelatin weight ratio ofbetween 0.5 and 100. This ratio is more preferably between 0.5 and 60,and in a particularly preferred manner, between 3 and 15. By way ofspecific examples, there may be mentioned ratios of 0.74, 1.66, 3.3,6.66, 13.4, 26.66 and 60. These various ratios correspond to mediaaccording to the invention containing from 10% to 90% by volume of acommercial plasmion solution and from 90% to 10% by volume of a humanserum albumin solution at 20%. Different compositions are represented byway of illustration in Table 2 below.

TABLE 2 Gelatin HSA Empirical Plasmion Weight HSA 20% Weight ratio % v(g/l) % v (g/l) HSA/gelatin 10 3 90 180 60 90 27 10 20 0.74 80 24 20 401.66 20 6 80 160 26.66 50 15 50 100 6.66 67 20 33 66 3.3 33 10 67 13413.4

In general, the medium according to the invention contains from 10 to90% by volume of plasmion respectively of human serum albumin solutionat 20%. The specific embodiments are media comprising between 20 and 80%of plasmion respectively of human serum albumin solution at 20%, morepreferably between 33 and 67% of plasmion respectively of human serumalbumin solution at 20%. Another specific embodiment of the inventionconsists in a mixture of plasmion and human serum albumin solution at20% in a volume to volume ratio of 50/50. Advantageously, theHSA/gelatin weight ratio is between 2 and 7. Particularly remarkableresults have been obtained with a ratio of about 3.

Moreover, additional components may be added to the media according tothe invention. In particular, biocompatible cell stabilizing agents maybe introduced, such as for example compounds of the glycerol family(glycine, glycerol, sucrose, glucose, and the like). These compounds arepresent in the media of the invention in quantities of less than 5% byweight. Preferably, the media according to the invention comprisebetween 0.5 and 5% by weight of glycine or glycerol.

The media according to the invention serve for the storage, preservationand freezing of biological material. Biological material is generallyunderstood to mean any material containing a genetic information, whichis self-reproducible or reproducible in a biological system. The saidbiological material may consist more particularly of cells or viralparticles or both. Among the cells which may be frozen, there may bementioned, for example, blood cells, bone marrow cells, cells producingviral particles (“packaging” lines), or genetically modified cells.

The viral particles more particularly relevant to the present inventionare those which may be used in gene therapy. A large number of virusesmay have their genome modified, on the one hand so that they lose theirability to multiply while retaining their infectivity, on the other handso as to insert into their genome a nucleic acid sequence of therapeuticinterest which will be expressed in the infected cells. Among theseviruses, there may be mentioned more particularly the adenoviruses, theAAVs, the retroviruses, the herpes viruses and the like.

The adenoviruses are among the most widely used viruses. Differentadenovirus serotypes, whose structure and properties vary somewhat, havebeen characterized. Among these serotypes, the use of type 2 or 5 humanadenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin (seeapplication WO 94/26914) is preferred in the context of gene therapy.Among the adenoviruses of animal origin which can be used, there may bementioned the adenoviruses of canine, bovine, murine, (example: MAV1,Beard et al., Virology 75 (1990) 81), ovine, porcine, avian or simian(example:SAV) origin. Preferably, the adenovirus of animal origin is acanine adenovirus, more preferably a CAV2 adenovirus [manhattan strainor A26/61 (ATCC VR-800) for example]. Preferably adenoviruses of humanor canine or mixed origin are used.

The defective adenoviruses comprise the ITRs, a sequence allowingencapsidation and a nucleic acid of interest. In the genome of theseadenoviruses, at least the E1 region is non-functional. The viral geneconsidered may be made non-functional by any technique known to personsskilled in the art, and especially by total suppression, substitution,partial deletion or addition of one or more bases in the gene(s)considered. Such modifications may be obtained in vitro (on isolatedDNA) or in situ, for example, by means of genetic engineeringtechniques, or alternatively by treatment by means of mutagenic agents.Other regions may also be modified, and especially the region E3(WO95/02697), E2 (WO94/28938), E4 (WO94/28152, WO94/12649, WO95/02697)and L5 (WO95/02697). It may also comprise a deletion in the E1 region atthe level of which the E4 region and the nucleic acid of therapeuticinterest are inserted (cf FR 94 13355).

The defective recombinant adenoviruses may be prepared by any techniqueknown to persons skilled in the art (Levrero et al., Gene 101 (1991)195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they maybe prepared by homologous recombination between an adenovirus and aplasmid carrying, inter alia, the DNA sequence of interest. Thehomologous recombination occurs after co-transfection of the saidadenoviruses and plasmid into an appropriate cell line. The cell lineused should preferably (i) be transformable by the said elements, and(ii), comprise the sequences capable of complementing the defectiveadenovirus genome part, preferably in integrated form in order to avoidthe risks of recombination.

Next, the adenoviruses which have multiplied are recovered and purifiedaccording to conventional molecular biology techniques, generally on acaesium chloride gradient.

As regards the adeno-associated viruses (AAV), they are DNA viruses of arelatively small size, which integrate into the genome of the cellswhich they infect, in a stable and site-specific manner. They arecapable of infecting a broad spectrum of cells, without inducing anyeffect on cell growth, morphology, or differentiation. Moreover, they donot appear to be involved in pathologies in man. The AAV genome has beencloned, sequenced and characterized. It comprises about 4,700 bases, andcontains, at each end, an inverted repeat region (ITR) of about 145bases, serving as replication origin for the virus. The rest of thegenome is divided into 2 essential regions carrying the encapsidationfunctions: the left part of the genome, which contains the rep geneinvolved in the viral replication and the expression of the viral genes;the right part of the genome, which contains the cap gene encoding thevirus capsid proteins.

The use of AAV-derived vectors for the transfer of genes in vitro and invivo has been described in the literature (see especially WO 5 91/18088;WO 93/09239; U.S. Pat. Nos. 4,797,368, 5,139,941, EP 488 528). Theseapplications describe different AVV-derived constructions, in which therep and/or cap genes are deleted and replaced by a gene of interest, andtheir use to transfer in vitro (on cells in culture) or in vivo(directly in an organism) the said gene of interest. The defectiverecombinant AAVs may be prepared by co-transfection, into a cell lineinfected by a human helper virus (for example an adenovirus), of aplasmid containing a nucleic acid sequence of interest bordered by twoAAV inverted repeat regions (ITR), and of a plasmid carrying the AAVencapsidation genes (rep and cap genes). A useable cell line is forexample the line 293. The recombinant AAVs produced are then purified byconventional techniques.

As regards the herpes viruses and the retroviruses, the construction ofrecombinant vectors has been widely described in the literature; seeespecially Breakfield et al., New Biologist 3 (1991) 203; EP 453242, EP178220, Bernstein et al. Genet. Eng. 7 (1985) 235; McCormick,BioTechnology 3 (1985) 689, and the like. In particular, theretroviruses are integrative viruses, selectively infecting dividingcells. They therefore constitute vectors of interest for cancerapplications. The genome of the retroviruses comprises essentially twoLTRs, an encapsidation sequence and three coding regions (gag, pol andenv). In the recombinant vectors derived from the retroviruses, the gag,pol and env genes are generally deleted, completely or in part, andreplaced by a heterologous nucleic acid sequence of interest. Thesevectors may be prepared from different types of retroviruses such asespecially MoMuLV (“murine moloney leukaemia virus”; also called MoMLV),MSV (“murine moloney sarcoma virus”), HaSV (“Harvey sarcoma virus”); SNV(“spleen necrosis virus”); RSV (“Rous sarcoma virus”) or Friend's virus.

To construct defective recombinant retroviruses comprising a nucleicacid of therapeutic interest, a plasmid comprising especially the LTRs,the encapsidation sequence and the said nucleic acid is constructed, andthen used to transfect a so-called encapsidation cell line, capable ofproviding in trans the retroviral functions deficient in the plasmid.Generally, the encapsidation lines are therefore capable of expressingthe gag, pol and env genes (cf below).

The generally recombinant viral particles (containing a nucleic acid ofinterest) and the defective viral particles (incapable of autonomousreplication) may be preserved in a medium according to the invention.Generally, the viral particles (adeno, AAV, retro and the like) are usedin purified form and then packaged in a medium according to theinvention for their preservation, preferably in frozen form. Generally,10⁴ to 10¹⁴ viral particles may be packaged in 1 ml of medium accordingto the invention, in a sterile container. Preferably, 10⁵ to 10¹⁰ viralparticles are used per ml of medium and, still more preferably, 10⁸ or10⁹.

The cell lines termed encapsidation (or packaging) lines which wereearlier referred to above are cell lines used for the production ofdefective recombinant viruses, in vitro or in vivo (after implantation).There may be mentioned, by way of example of a line for the productionof adenoviruses, the human embryonic kidney line 293 (Graham et al., J.Gen. Virol. 36 (1977) 59) which contains especially, integrated into itsgenome, the left part of the genome of an Ad5 adenovirus (12%) or linescapable of complementing the E1 and E4 functions as described especiallyin applications Nos. WO 94/26914 and WO 95/02697. There may also bementioned encapsidation lines which have been described for theproduction of retroviruses or herpes viruses, and especially the linePA317 (U.S. Pat. No. 4,861,719); the line PsiCRIP (WO 90/02806) and theline GP+envAm−12 (WO 89/07150) or recombinant retrovirus-producingderived lines such as the line M11 (WO 94/13824). As illustrated in theexamples, the medium of the invention is particularly adapted to thepreservation of viral particle-producing cells.

Another type of biological material which may be advantageouslypreserved in a medium according to the invention consists of geneticallymodified cells. The genetically modified cells are cells, intendedespecially for gene therapy, into which a nucleic acid sequence ofinterest has been introduced. Over the past few years, the number ofthese cells has continued to grow and there may be mentioned, by way ofexamples, the haematopoietic stem cells (WO 88/08450, WO 93/20195, WO93/09815, WO 93/11230), the endothelial cells (WO 89/05345, WO 90/06757,WO 92/09222), the myoblasts (WO 93/03768, WO 93/24151, WO 94/01129), thefibroblasts (U.S. Pat. No. 5,219,740, WO 89/02468, WO 93/07906), thehepatocytes (WO 89/07136, WO 92/12242, WO 93/03142), the astrocytes (WO94/01135), the neuroblasts (WO 94/10292, WO 94/16059), the keratinocytes(WO 94/11011), the macrophages (FR 93/10222). These cells generallypossess the capacity to produce a therapeutic product of interest andmay be implanted in vivo. Among the blood cells, there may be mentionedthe erythrocytes, the neutrophilic, basophilic and eosinophilicgranulocytes, the B and T lymphocytes, especially the CD4 lymphocytes,the cytotoxic lymphocytes (CD8 CTL), the tumour infiltrating lymphocytes(TIL) and the LAKs, the monocytes and macrophages, the dendritic cells,the megakaryocytes and the platelets. These cells may also begenetically modified in order to acquire new therapeutic properties.

The medium according to the invention may, in addition, be used for thepreservation of primary cell cultures, and of tumour cells or biopsiesof tumours. This type of material is currently under study in clinicaltrials of immuniadoptive therapy in which a tumour is removed from apatient, treated with different immunopotentiating agents (introductionof genetic material expressing lymphokines or tumour antigens) and thenreadministered to the patient. One of the difficult steps lies in thepreservation of the cells removed from the patient or modified beforereinfusion. The medium of the invention advantageously allows a goodpreservation of these materials with a high viability.

The use of a medium according to the invention makes it possible topreserve these cells and to inject them directly into an organism,without a centrifugation or washing stage, with a good viability andwithout affecting their capacity to produce therapeutic proteins orviruses, where appropriate.

To this end, the present invention also relates to preparationscontaining the preservation medium according to the invention andbiological material, as well as to a process for the storage ofbiological material. The said biological material may be packageddirectly in a medium according to the invention. As regards cells, theseare advantageously previously freed of their culture medium (for examplecentrifuged, harvested and washed in a buffer solution), before beingpackaged in a medium according to the invention. As regardsproliferative cells, they are preferably used at sub-confluence, at theexponential growth phase. As indicated in the examples, it is underthese conditions that the best viability results may be obtained. It ispossible, however, to use cells at confluence or post-confluence.Generally, 10⁵-10⁹ cells are packaged per ml of medium and, morepreferably, 10⁶-10⁸. In the case of adherent cells, the cells arepreviously detached by conventional treatment, suspended and thenpackaged in a medium of the invention. The treatment used to detach thecells may be an enzymatic treatment (trypsin for example), a chemicaltreatment (detergent) or a mechanical treatment. In the case of achemical or enzymatic treatment, the cells are then centrifuged andwashed in order to remove the enzyme or the detergent, before freezing.Generally, the viability of the cells is controlled before freezing, aswell as their sterility. As regards viruses, these are previouslypurified as indicated above (centrifugation on a caesium chloridegradient for example, chromatographies and the like). They may bepackaged at the rate of 10⁴ to 10¹⁴ particles per ml, preferably 10⁵ to10¹⁰. The biological material may then be packaged in the mediumaccording to the invention, in an appropriate container. It may be anampoule, a tube, especially a cryotube, a bag, a vial, a flask and thelike. The container is previously sterilized and the packagingoperations are performed under sterile conditions.

A medium according to the invention allows the freezing and thawing ofbiological material under conditions of high viability. The mediaaccording to the invention may, in particular, allow the freezing ofbiological material at temperatures of between −200 and −4 degreesCelsius. The material may be preserved especially in liquid nitrogen orat higher temperatures, for a period which is sufficiently long toensure the preservation of an industrial stock (up to one year forexample). The percentage of viable cells after thawing is defined as thenumber of live cells divided by the total number of cells multiplied byone hundred. This percentage viability in a medium according to theinvention is advantageously greater than 50%. Preferably, thispercentage is greater than 60%. Most preferably, this percentage isgreater than 70%.

Other advantages of the present invention will appear on reading thefollowing examples which should be considered as illustrative andnon-limiting.

Materials and Methods

Test of Cell Viability

The viability of the cells was determined by the trypan blue technique.Trypan blue is a dye which penetrates only into dead cells. When thevery regular grid of a Malassez cell or of a Kova slide is observed, thecells which are in the small squares of the grid as a whole arecontained in a very precise volume: 1 μl. These are the cells which arecounted, while observing the following rules:

The cells which are on the outer limits of the grid are counted only ifthey are on the upper and left edges (green) or on the lower and rightedges (red);

For the counting to be significant, count two counting chambers andcalculate the mean.

The number of dead cells corresponds to the number of blue cells. Thenumber of viable cells corresponds to the number of refringent whitecells. The viability is expressed by the ratio of the number of viablecells to the number of viable cells plus the number of dead cells.

It is understood that other techniques for counting or determining cellviability may be used.

Cells Used

The cells used in the examples are genetically modified fibroblastscapable of producing viral particles. They are more precisely of thecell line M11 deposited at the Collection Nationale de Culture deMicroorganismes, under the reference I-1278. This line derives from thePsiCRIP cells.

Equivalent cells are for example the cells of the Am12 line (WO89/07150). It is understood that the process described is directlyapplicable to other cells, especially of human origin, whether they areprimary cultures or established lines.

Albumin

The albumin used in the context of the examples is marketed by thecompany Armour under the reference Albumin A or human plasma Albumin20%. It is understood that any other source of albumin may be used.

Plasmion

Plasmion is of commercial origin (Roger Bellon, France).

EXAMPLES Example 1 Procedure for Freezing the Biological Material

For the freezing of cells, it is preferable to use subconfluent cells atthe exponential growth phase. Moreover, to improve the monitoring of thebiological material, the distribution and freezing, in cryotubes, of thecell suspension after bringing into contact with the freezing mediumaccording to the invention are carried out as rapidly as possible.Finally, the manipulations are performed under sterile conditions (forexample in a laminar flow cabinet).

This example describes more particularly a procedure for freezing cells(genetically modified cells, blood cells, virus producing cells and thelike). It is understood that this procedure may be adapted by personsskilled in the art to the freezing of virus or other material.

The culture flasks containing the cells to be frozen are taken out ofthe incubator, and placed under a microscope, so as to check theappearance of the culture. If one or more flasks have a non-conformingappearance (detached cells, confluent cells, cloudy culture,non-refringent cells, impaired flask, and the like), it is not used forthe freezing.

The conforming flasks are then transferred into a laminar flow cabinet.When the cells are adhering cells, they are treated so as to detach themand/or dissolve the aggregates. For that, it is possible to use trypsinor any other dissociating medium (detergent, and the like). The cellsuspensions are then combined in a sterile flask and gently homogenizedby pipetting. An aliquot of 1 ml is removed for the counting. If theviability of the culture is less than 80%, the cell suspension isrejected.

The suspension is then distributed, equally (for example using apipette) into an even number of centrifugation tubes and thencentrifuged for 10 minutes at 400 g.

The containers for freezing using alcohol are transferred to thecabinet, as well as a flask of freezing medium at +4° C. The volume offreezing medium necessary to obtain a cell concentration of 10⁷ viablecells per ml is then placed in a sterile flask.

After centrifugation, the supernatant is removed and then the pelletsare taken up in aliquots of cold freezing medium. The suspensions arehomogenized and taken up in the flask containing the freezing medium,and again homogenized. A sample is collected for the test of sterility.

The cell suspension is then distributed in the cryotubes, which are thenplaced immediately in the container for freezing using alcohol. Thecontainers are transferred for 1 hour at +4° C. and then placed in achamber at −80° C. for at least 12 h, preferably 24 h. At least 24 hafter freezing, the ampoules are removed from containers with alcoholand stored in a container with liquid nitrogen.

Example 2 Procedure for Thawing the Biological Material

A. Products used

Trypan blue

Sterile phosphate buffer (PBS), pH 7.2

Ethanol 70%

Culture medium

Foetal calf serum (FCS)

Sterile pot of water at 37°±0.5° C. (50 to 200 ml)

B. Procedure

The freezing is advantageously performed under sterile conditions, forexample in a safety cabinet.

In a 50 ml sterile tube, prepare a volume of culture medium at 20% FCScorresponding to {fraction (9/10)} of the volume of the batch (of theampoule) to be thawed (thawing medium). Thus, 7 ml of culture mediumplus 2 ml of FCS are prepared in the case of a 1 ml ampoule.

The ampoule to be thawed is dipped into the pot of sterile water atabout 37 degrees Celsius, without submerging it, with gentle stirringuntil the ice completely disappears. Rapidly wipe the ampoule with 70%ethanol. Pipette the contents of the ampoule and transfer it into thetube containing the thawing medium and then, without changing thepipette, reaspirate an identical volume and rinse the ampoule once.Gently homogenize the suspension and collect 1 ml in a sterile tube forcounting. Depending on the counting result, the cell suspension istransferred into a culture flask, diluting with the quantity of thawingmedium necessary to obtain an initial cell concentration of between 2.0and 3.0×10⁵ viable cells per millilitre of culture.

To allow the cell viability after thawing to be monitored over time, theculture flask is then placed in the CO₂ incubator at 37° C.±0.5; or inthe case of HEPES buffered medium, in a hot chamber at 37° C.+0.5.

In the case of therapeutic applications, only one aliquot of thecontents of the ampoule is used for the test of cell viability andsterility. If there is conformity, the contents of the thawed ampoulemay then be directly injected.

Example 3 Study of the Percentage Viability After Freezing and Thawingof Retrovirus-producing Cells in Different Media According to theInvention.

The cells used in this example are recombinant retrovirus producingcells of the M11 line. The cells are used at the exponential growthphase. The cells were frozen according to the general proceduredescribed in Example 1. For this, the cells were detached from the dishwith the dissociating medium (1×PBS: 0.02% EDTA): 3 ml per T75 dish; 5ml per T160 or T225 dish. The cells are incubated for 5 min in thismedium, with gentle stirring, and then recovered in a Falcon tube. Analiquot is used to count the cells. The suspension is then centrifugedfor 5 min at 1000 rpm at 20° C. The cells are then distributed intofreezing ampoules containing 1 ml of medium according to the invention,at the rate of 10⁷ cells per ml. The different media tested aredescribed below (Table 3). The ampoules are frozen according to theprocedure described in Example 1. At a determined date, the ampoules arethawed according to the procedure described in Example 2, and the cellsuspension is transferred into culture flasks 37° C., 5% CO₂. The cellviability is determined as indicated in Materials and Methods. Theresults obtained are presented in the following tables 4-10. Theyclearly show a high percentage viability in the presence of a medium ofthe invention. Thus, more than 70% of cells are viable for certain mediaof the invention. In general, the cell viability is always greater than50%. It should be noted that no single component of the media of theinvention makes it possible to obtain a stability greater than 25%.

A control of viability and of revival in culture was carried out onbatches after being frozen for 7 months. Thus, 4 ampoules of cellsfrozen in a solution of Plasmion 67%/HSA 33% (see Example 1) were thawedafter 7 months according to the protocol of Example 2. The four ampouleswere cultured in a 75 cm² flask. The viability and revival in culturewere determined and are presented in Table 11.

These results show a high percentage viability and a good revival of thecells in culture. The cells exhibit normal adherence and a normalrefringent appearance. The passages P1 and P2 (175 cm²) were carried outunder normal culture conditions.

Example 4 Improvement of the Revival in Culture

In order to study the revival in culture of the cells treated accordingto the invention, tests were carried out on different media supplementedwith cell stabilizing agents, and in particular with differentconcentrations of glycerol.

4.1. Preparations of the Media

A [lacuna] is prepared according to the procedure described above, andthen glycerol is next added at the concentrations indicated in the tablebelow, or, as a control, DMSO. Briefly, for a volume of 10 ml of afreezing medium at 67% plasmion, 33% HSA and 2.5% glycerol, theconcentrations by volume of the components are:

HSA/Plasmion medium: 9.75 ml

Glycerol: 0.25 ml or 0.312 g of glycerol

10 ml of HSA/Plasmion→6.7 ml of Plasmion

9.75 ml of HSA/Plasmion medium→6.53 ml of plasmion

10 ml of HSA/Plasmion medium→3.3 ml of HSA

9.75 ml of HSA/Plasmion medium→3.21 ml of HSA

TABLE 12 Composition of the supplemented media PLASMION HSA DMSOGLYCEROL MEDIA Q:10 ml (ml) (ml) (ml) (ml) HP + 5% DMSO 6.3 3.13 0.5HP + 2.5% DMSO 6.53 3.21 0.25 HP + 1% DMSO 6.63 3.26 0.1 HP + 10%GLYCEROL 6.03 2.97 1 (1.25 g) HP + 5% GLYCEROL 6.3 3.13 0.5 (0.625 g)HP + 2.5% GLYCEROL 6.53 3.21 0.25 (0.312 g) HP + 1% GLYCEROL 6.63 3.260.1 (0.125 g)

4.2. Study of Cell Viability

The freezing was carried out under the conditions described inExample 1. The results obtained after thawing on day 7 are presented inTable 13. They show that, in the presence of glycerol in a proportion ofless than about 5%, the mean cell viability observed after thawing isgreater than 70%. In particular, a mean viability of 84% is observed inthe presence of 1% glycerol.

4.3. Study of the Revival in Culture

The revival in culture is a variable characterizing the state ofpoliferative cells after thawing. It is estimated, in this example, bythe time necessary for the cells to reach confluence. To determine thisparameter, after thawing, the cell concentration is adjusted to between2.5×10⁵ and 3.5×10⁵ C/ml then the cells are maintained in culture. Theresults obtained are presented in Table 14. They show that the revivalin culture occurs more rapidly when the cells have been frozen in amedium supplemented with glycerol (3.5 to 4 days) than in a mediumwithout glycerol (4 to 5 days). These results also show that the cellstabilizing agent is advantageously introduced in an amount of 0.5 to2.5%.

Example 5 Programmed Freezing Tests

Programmed freezing tests were carried out in order to determine if abetter control of the freezing parameters could influence the quality ofthe biological material. In particular, a freezing procedure which makesit possible to avoid superfusion of the medium was tested. To do this,freezing tests assisted by a liquid nitrogen freezer Kryosave planarfrom Flobio were carried out, in comparison with freezing tests usingalcohol (isopropanol) according to Example 1. The freezing is performedin a freezing medium at 67% plasmion, 33% HSA and 2.5% or 1% glycerol.The advantage of this system is to carry out a rapid freezing of thecells (or of the material), and a controlled decrease in the temperatureof the cells and of the medium, which makes it possible to avoid themelting point of any frozen medium. For that, the melting point isdetermined beforehand by observing, during freezing using alcohol, itsposition in terms of temperature and over time. In this example, thetemperature was reduced to −40° C. over 10 minutes from −8° C., becausethe melting point for the material used was at this level and the mediumwas rising towards 0° C. After freezing on day 7, the cell viability andthe revival in culture after 24 hours are determined. The resultsobtained are presented in Tables 15 and 16.

These results clearly show a high cell viability, in all the freezingschemes and media of the invention tested. In addition, they show a goodrevival of the cells in culture, in particular for media comprising 1%glycerol. In this case, indeed, a yield greater than 70% is observed 24hours after thawing. Moreover, in the case of a controlled programmedfreezing (Kryosave), the cell layer observed after 24 hours is good anda very limited number of dead cells appears.

TABLE 3 Composition and preparation of the media H/G Prepar- 20% HSA g/lg/l Weight ation Medium Solution Plasmion HSA Gelatin ratio (10 ml)10H/90P 10% v 90% v 20 27 0.740 1 ml HSA 9 ml P 20H/60P 20% v 80% v 4027 1.666 2 ml HSA 6 ml P 30H/70P 30% v 70% v 60 21 2.657 3 ml HSA 7 ml P40H/60P 40% v 60% v 80 18 4.444 4 ml HSA 6 ml P 50H/50P 50% v 50% v 10015 6.666 5 ml HSA 5 ml P 60H/40P 60% v 40% v 120 12 10 6 ml HSA 4 ml P70H/30P 70% v 30% v 140 9 15.555 7 ml HSA 3 ml P 60H/20P 80% v 20% v 1606 26.666 8 ml HSA 2 ml P 90H/10P 90% v 10% v 160 3 60 9 ml HSA 1 ml P

TABLE 4 Experiment #1 - Thawing on day 5 Number of cells per % viabilityampoule Freezing medium on thawing (*10⁷) Culture 67H/33P 50.8 0.975 Yes50H/50P 63.2 0.51 Yes 33H/67P 71.4 0.42 Yes H/P + Glycine 5% 49.4 0.51Yes H/P + Glycine 1% 54.8 0.54 Yes

TABLE 5 Experiment #1 - Thawing on day 6 Number of cells per % viabilityampoule Freezing medium on thawing (*10⁷) Culture 67H/33P 76.2 0.69 Yes50H/50P 65.9 0.69 Yes 33H/67P 55.1 0.76 Yes H/P + Glycine 5% 63.2 0.21Yes H/P + Glycine 1% 55.6 0.25 Yes

TABLE 6 Experiment #2 - Thawing on day XX Number of cells per %viability ampoule Freezing medium on thawing (*10⁶) Culture 67H/33P 55.210.4 Yes 43.2 13.2 Yes 50H/50P 60.9 28.8 Yes 63.7 26 Yes 70.5 12.1 Yes71.7 9 Yes 33H/67P 73.5 12.8 Yes 82 8.55 Yes

TABLE 7 Experiment #3 - Thawing on day 5 Number of cells per % viabilityampoule Freezing medium on thawing (*10⁷) Culture 10H/90P nd nd nd20H/80P 53.4 1.10 Yes 30H/70P 54.4 0.86 Yes 40H/60P 53.5 1.29 Yes50H/50P 46.3 1.00 Yes 60H/40P 48   1.13 Yes 70H/30P nd nd nd 80H/20P59.2 1.14 Yes 90H/20P 61.7 0.90 Yes

TABLE 8 Experiment #3 - Thawing on day 13 Number of cells per %viability ampoule Freezing medium on thawing (*10⁷) Culture 10H/90P 49.20.94 Yes 20H/80P 54.5 1.12 Yes 30H/70P nd nd nd 40H/60P 50.6 1.20 Yes50H/50P 58   0.73 Yes 60H/40P 49.4 1.20 Yes 70H/30P 54.8 0.90 Yes80H/20P 64.7 0.99 Yes 90H/20P 50   0.79 Yes

TABLE 9 Experiment #4 - Thawing on day 8 Number of cells per % viabilityampoule Freezing medium on thawing (*10⁷) Culture 50H/50P 61.8 0.83 Yes30H/70P 68 0.75 Yes 70H/30P 59.2 0.74 Yes 100P 21.9 0.96 No

TABLE 10 Experiment #4 - Thawing on day 19 Number of cells per %viability ampoule Freezing medium on thawing (*10⁷) Culture 50H/50P 77.30.66 Yes 30H/70P 61.2 0.735 Yes 70H/30P 62.7 0.85 Yes 100P 6.5 0.69 No

TABLE 11 Viability and revival in culture after thawing at 7 monthsAdherent Adherent Total Number Adherent cells cells no. of of live cells72 h 120 h Yield Viability cells cells 24 h P1 P2 24 h Amp. 1 79.8% 3.1310⁷ 2.5 10⁷ 1.36 10⁷ 54.4% Amp. 2 74.5% 3.1 10⁷ 2.31 10⁷ 1.66 10⁷ 3.4810⁷ Amp. 3 79% 2.72 10⁷ 2.17 10⁷ 1.35 10⁷ 62.2% Amp. 4 76.6% 3.25 10⁷2.49 10⁷ NA NA NA

TABLE 13 Glycerol-supplemented media Mean V1 V2 V3 viability HP + 10%Glycerol 41% 50% 48.5% 46.5% HP + 5% Glycerol 70% 70% 64% 68% HP + 2.5%Glycerol 80.5% 80% 79% 79.8% HP + 1% Glycerol 84% 82% 87% 84% HP 78% 75%73% 75% HP + 5% DMSO 81% 91% 93% 88% HP + 2.5% DMSO 94% 89% 92% 91% HP +1% DMSO 93% 92% 89% 91%

TABLE 14 Study of the revival in culture Confluence 1 Confluence 2Confluence 3 75 cm² 75 cm² 25 cm² HP + 10% Glycerol no sub- no sub- nosub- culturing culturing culturing HP + 5% Glycerol 4 4 3.5 HP + 2.5%Glycerol 4 4 3.5 HP + 1% Glycerol 4 4 3.5 HP 5 5 4  

TABLE 15 Programmed freezing tests in HP + 1% Glycerol medium Cell Totallive Adh. cells Dead cells Viability conc./ml cells 24 h in susp. YieldKryosave 1 88% 7.3 × 10⁵ 2.19 × 10⁷ 1.74 × 10⁷ 4.95 × 10⁶ 79% Cisopropanol 1 77% 6 × 10⁵ 1.81 × 10⁷ 1.34 × 10⁷ 1.32 × 10⁷ 74% Kryosave2 83% 8.4 × 10⁵ 2.53 × 10⁷ 1.95 × 10⁷ 3.3 × 10⁶ 77% C isopropanol 281.5% 8 × 10⁵ 2.41 × 10⁷ 1.02 × 10⁷ 1.12 × 10⁷ 42% All the cells in eachampoule are placed in culture in 75 cm²flasks

TABLE 16 Programmed freezing tests in HP + 2.5% Glycerol medium CellTotal live Adh. cells Dead cells Viability conc./ml cells 24 h in susp.Yield Kryosave 1 85% 1.54 × 10⁶ 4.62 × 10⁷ 2.07 × 10⁷ 5.1 × 10⁶ 45%Isopropanol 1 82% 1.12 × 10⁶ 3.36 × 10⁷ 1.03 × 10⁷ 6.9 × 10⁶ 30%Kryosave 2 78% 1.76 × 10⁶ 5.28 × 10⁷ 2.9 × 10⁷ 3.9 × 10⁶ 55% Isopropanol2 69% 9.7 × 10⁵ 2.91 × 10⁷ 1.45 × 10⁷ 5.25 × 10⁶ 50%

What is claimed is:
 1. A medium for preservation of frozen biologicalmaterial, wherein the medium is directly injectible into an organism andconsists of an isotonic saline solution, modified fluid gelatin andhuman serum albumin, and wherein the medium lacks a toxic agent.
 2. Themedium according to claim 1, wherein the isotonic saline solution isisotonic with plasma.
 3. The medium according to claim 2, wherein theisotonic saline solution consists of sodium chloride, potassiumchloride, magnesium chloride, and sodium lactate.
 4. The mediumaccording to claim 3, wherein the isotonic saline solution consists of 2to 5 g/l of sodium chloride, 0.05 to 0.5 g/l of potassium chloride, 0.05to 0.2 g/l of magnesium chloride and 0.5 to 4 g/l of sodium lactate. 5.The medium according to claim 2, wherein the isotonic saline solutionconsists of sodium chloride, potassium chloride, calcium chloride andsodium lactate.
 6. The medium according to claim 5, wherein the isotonicsaline solution is Ringer's lactate.
 7. The medium according to claim 1,wherein the modified fluid gelatin is a chemically modified collagenhydrolysis product.
 8. The medium according to claim 7, wherein thechemically modified collagen hydrolysis product has an average molecularweight of between 10 kD and 100 kD.
 9. The medium according to claim 8,wherein the chemically modified and pharmaceutically acceptable collagenhydrolysis product has an average molecular weight of between 15 kD and40 kD.
 10. The medium according to claim 7, wherein the modified fluidgelatin is chemically modified by reacting collagen with succinic,citraconic, itaconic, aconitic, or maleic anhydride.
 11. The mediumaccording to once claim 1, wherein the human serum albumin is of plasmaorigin.
 12. The medium according to claim 1, wherein the medium has ahuman serum albumin (grams/liter)/gelatin (grams/liter) ratio of between0.5 and
 100. 13. The medium according to claim 12, wherein the humanserum albumin/gelatin ratio is between 0.74 and
 60. 14. The mediumaccording to claim 12, wherein the human serum albumin/gelatin ratio isequal to 0.74, 1.66, 3.3, 6.66, 13.4, 26.66, or
 60. 15. A compositioncomprising a population of genetically modified cells and the mediumaccording to claim
 1. 16. The composition of claim 15, wherein the cellsare blood cells.
 17. The composition according to claim 16 wherein theblood cells are platelets.
 18. A composition comprising a population ofbone marrow cells and the medium according to claim
 1. 19. A compositioncomprising viral particles and the medium according to claim
 1. 20. Thecomposition according to claim 19, wherein the viral particles arereplication defective recombinant viral particles.
 21. A compositioncomprising a population of viral particle-producing cells and the mediumaccording to claim
 1. 22. A process for storing isolated cells or viralparticles comprising suspending the isolated cells or viral particles ina medium according to claim 1, and freezing the suspension.
 23. Theprocess according to claim 22, wherein the biological material isisolated cells and wherein a percentage of cell viability after thawingis greater than or equal to 50%.
 24. The process according to claim 23,wherein the percentage of cell viability after thawing is greater thanor equal to 60%.
 25. The process according to claim 24, wherein thepercentage of cell viability after freezing and thawing is greater thanor equal to 70%.
 26. A medium for preservation of frozen biologicalmaterial, wherein the medium is directly injectible into an organism andconsists of an isotonic saline solution, modified fluid gelatin, humanserum albumin and between 0.5% and 5% by weight of a biocompatible cellstabilizing agent, and wherein the medium lacks a toxic agent.
 27. Themedium according to claim 26, wherein the cell stabilizing agent isselected from the group consisting of glycine, glycerol, sucrose, andglucose.
 28. A frozen biological composition comprising an isotonicsaline solution, modified fluid gelatin, human serum albumin and atleast one of animal cells or viral particles; wherein the percentage ofviability of the cells or viral particles is greater than or equal to50% upon thawing of the frozen biological composition, and the frozenbiological composition lacks any agent which would be toxic to anorganism injected with the thawed biological composition.
 29. The frozenbiological composition according to claim 28, wherein the isotonicsaline solution is isotonic with plasma.
 30. The frozen biologicalcomposition according to claim 29, wherein the isotonic saline solutioncomprises sodium chloride, potassium chloride, magnesium chloride andsodium lactate.
 31. The frozen biological composition according to claim30, wherein the isotonic saline solution comprises 2 to 5 g/l of sodiumchloride, 0.05 to 0.5 g/l of potassium chloride, 0.05 to 0.2 g/l ofmagnesium chloride and 0.5 to 4 g/l of sodium lactate.
 32. The frozenbiological composition according to claim 29, wherein the isotonicsaline solution comprises sodium chloride, potassium chloride, calciumchloride and sodium lactate.
 33. The frozen biological compositionaccording to claim 32, wherein the isotonic saline solution is Ringer'slactate.
 34. The frozen biological composition according to claim 28,wherein the modified fluid gelatin is a chemically modified collagenhydrolysis product.
 35. The frozen biological composition according toclaim 34, wherein the chemically modified collagen hydrolysis producthas an average molecular weight of between 10 kD and 100 kD.
 36. Thefrozen biological composition according to claim 34, wherein themodified fluid gelatin is chemically modified by reacting collagen withsuccinic, citraconic, itaconic, aconitic or maleic anhydride.
 37. Thefrozen biological composition according to claim 28, wherein the humanserum albumin is of plasma origin.
 38. The frozen biological compositionaccording to claim 28, having a human serum albumin to modified fluidgelatin ratio of between 0.5 and 100, wherein both the human serumalbumin and modified fluid gelatin are measured in grams per liter. 39.The frozen biological composition according to claim 38, wherein thehuman serum albumin to modified fluid gelatin ratio is between 0.74 and60.
 40. The frozen biological composition according to claim 38, whereinthe human serum albumin to modified fluid gelatin ratio is selected fromthe group consisting of 0.74, 1.66, 3.3, 6.66, 13.4, 26.66 and
 60. 41.The frozen biological composition according to claim 28, furthercomprising between 0.5% and 5% by weight of a biocompatible cellstabilizing agent.
 42. The frozen biological composition according toclaim 41, wherein the biocompatible cell stabilizing agent is selectedfrom the group consisting of glycine, glycerol, sucrose and glucose. 43.The frozen biological composition according to claim 28, containinggenetically modified cells.
 44. The frozen biological compositionaccording to claim 28, containing blood cells.
 45. The frozen biologicalcomposition according to claim 44, wherein the blood cells areplatelets.
 46. The frozen biological composition according to claim 28,containing bone marrow cells.
 47. The frozen biological compositionaccording to claim 28, containing replication defective recombinantviral particles.